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【目的】克隆TaPrx基因并对其在小麦与条锈菌互作中的功能进行初步分析。【方法】利用PCR方法结合RACE技术在cDNA文库中筛选得到TaPrx基因的全长序列并进行生物信息学分析,然后克隆至pET-32a(+),转化E.coliBL21(DE3)后用IPTG进行诱导表达。通过Real-timeRT-PCR进行表达模式分析。【结果】得到TaPrx基因的全长序列688bp,ORF489bp,编码162个氨基酸残基,分子量17.36kD,等电点5.32,含一个保守的半胱氨酸残基(Cys),不含信号肽及跨膜结构域,亚细胞定位94%的可能性在细胞质。TaPrx融合蛋白分子量38kD,最佳IPTG诱导浓度0.05mmol·L-1,20℃诱导20h可得到最大量的融合蛋白。Real-timeRT-PCR分析表明TaPrx基因在小麦与条锈菌的亲和与非亲和互作中均受诱导表达,分别在接种后24h、18h达到表达高峰。【结论】获得了TaPrx基因特异性的多克隆抗体;TaPrx基因受条锈菌诱导表达,可能参与了小麦与条锈菌互作。但是否参与了小麦受条锈菌侵染后产生的ROS的清除与调节仍需进一步验证。
【Objective】 The objective of this study was to clone the TaPrx gene and analyze its function in the interaction between wheat and stripe rust. 【Method】 The full-length sequence of TaPrx gene was screened by bioinformatics analysis in cDNA library by PCR and RACE technique. The full-length TaPrx gene was cloned into pET-32a (+), transformed into E.coli BL21 (DE3) and induced by IPTG expression. Expression pattern analysis was performed by Real-time RT-PCR. 【Result】 The full-length sequence of TaPrx gene was 688bp in length and ORF489bp, encoding 162 amino acid residues with a molecular weight of 17.36kD and an isoelectric point of 5.32. A conserved cysteine residue (Cys) Membrane domain, 94% chance of subcellular localization in the cytoplasm. TaPrx fusion protein molecular weight of 38kD, the best IPTG induced concentration of 0.05mmol·L-1, 20 ℃ induced 20h to obtain the maximum amount of fusion protein. Real-time RT-PCR analysis showed that the TaPrx gene was induced in the affinity and non-affinity interaction between wheat and stripe rust, respectively, and reached the peak at 24h and 18h after inoculation. 【Conclusion】 The TaPrx gene-specific polyclonal antibody was obtained. The TaPrx gene was induced by stripe rust, which may be involved in the interaction between wheat and stripe rust. However, whether the involvement of wheat stripe rust infection generated ROS removal and regulation still need further verification.